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(Received for publication, January 17, 1996; and in revised form, February 6,
1996) From the
Several lines of evidence indicate that calreticulin has
lectin-like properties. As a molecular chaperone, calreticulin binds
preferentially to nascent glycoproteins via their immature
carbohydrates; this property closely resembles that seen for calnexin,
a chaperone with extensive molecular identity to calreticulin. A cell
surface form of calreticulin also exhibits lectin-like properties,
binding specific oligomannosides including those covalently linked to
laminin. In the present study we examined the interaction between
calreticulin and laminin by means of surface plasmon resonance. The
results show that calreticulin specifically binds to glycosylated
laminin but fails to specifically bind tunicamycin-derived
unglycosylated laminin or bovine serum albumin. Calreticulin binding to
glycosylated laminin requires calcium and is abolished in the presence
of EDTA. Scatchard analysis of binding yields an apparent association
constant, K Calreticulin is found in many different locations in various
eukaryotic cells, including the lumen of the endoplasmic reticulum
(ER), ( These
emerging lines of evidence describe lectin-like properties of
intralumenal and cell surface calreticulin. Ligand binding studies
indicate that intralumenal calnexin preferentially recognizes the
immature glycosyl structure,
Glc Calreticulin was kindly provided by R. Freedman; it had been
purified from bovine liver ER(2) . Glycosylated laminin was
purified from mouse Engelbreth-Holm-Swarm tumor while unglycosylated
laminin was purified from a mouse cell line incubated in
tunicamycin(15) . Tunicamycin-derived laminin lacks detectable N-linked carbohydrates, and its protein molecular structure
appears virtually identical to glycosylated laminin(15) . Binding analysis of the interaction between calreticulin and laminin
was performed on either a BIAcore or BIAcore 2000 biosensor (Pharmacia
Biosensor, Uppsala, Sweden) using contemporary technology(14) .
Experiments were performed at 25 °C in 10 mM HEPES-buffered saline, 150 mM NaCl, and 0.005% surfactant
P20 (Pharmacia) either with calcium (2 mM CaCl
Figure 1:
Binding of calreticulin in the presence
of calcium. In separate experiments, calreticulin solution ranging from
0.5
Figure 2:
Calreticulin binding to laminin.
Calreticulin binding to glycosylated and unglycosylated laminin was
measured in the presence of calcium or EDTA. RU, resonance
units.
Figure 3:
Scatchard analysis of calreticulin binding
to glycosylated laminin in the presence of calcium. RU,
resonance units.
Figure 4:
Binding of calreticulin in the presence of
EDTA. Titration was the same as described for Fig. 1. RU, resonance units.
The objective of this study was to further explore
interactions between calreticulin and the N-linked
carbohydrates of laminin. Conceivably, intralumenal glycosylated
laminin chains and/or assembled laminin molecules may transiently bind
to molecular chaperones, including those which recognize glycoproteins.
The present results provide a firm basis for potential ER intralumenal
binding of glycosylated laminin molecules to calreticulin. Presumably,
individual laminin subunits bind to calreticulin, followed by laminin
molecular assembly, perhaps while the subunits are still complexed to
the chaperone. Notably, laminin synthesized in the presence of
tunicamycin fails to be secreted(15) , perhaps reflecting the
inability of certain chaperones such as calreticulin to properly
interact with the unglycosylated protein. Lectins require a suitable
cation, often calcium, for sustaining their carbohydrate binding
properties. Oligomannosides have been specifically implicated in cell
surface calreticulin binding to laminin(7) . The present
results bolster the interpretation that calreticulin has lectin-like
activity by demonstrating that it binds to glycosylated laminin in the
presence of calcium while EDTA abolishes such binding. Calreticulin
fails to bind to unglycosylated laminin, further substantiating its
lectin-like properties. Intralumenal calreticulin binds nascent
transferrin(4) , a glycoprotein, and appears to interact with
nascent myeloperoxidase via that glycoprotein's N-linked
oligomannosides(3) , thereby resembling the binding of calnexin
to nascent glycoproteins(20) . Presumably, both intralumenal
calreticulin and calnexin rely upon calcium to support their
lectin-like activities. Binding of carbohydrate ligands to both
plant (24) and animal lectins (25) has been evaluated
by surface plasmon resonance. Association on rates ranging from 1.63
In mouse
melanoma cells the calreticulin-laminin complex itself may reach the
cell surface, accounting for calreticulin appearance on the surface (9) and consistent with the observation that these cells
produce and release laminin(21) . A precedent for postulating
such a pathway is that intralumenal calnexin, complexed to antigen
receptor proteins, reaches the surface of immature
thymocytes(22) . This complex transits to the thymocyte surface
from the ER, due to impairment of internal recycling of calnexin. The
authors speculate that surface calnexin may mediate cell-cell
lectin-dependent interactions and may also generate intracellular
signals. It is already clear that surface calreticulin recognizes
laminin (9) and fibrinogen (23) ; such recognition
leads to specific cellular responses in each instance. Mouse melanoma
cells, adherent to laminin, will spread once their surface calreticulin
binds to a suitable oligomannoside(7) . Human fetal fibroblasts
bind the fibrinogen B These studies measured the binding affinity
of bovine ER-derived calreticulin and murine laminin. Given that
calreticulin structure is highly conserved (18) and that the
oligomannoside N-linked structures are virtually
species-independent, it is not surprising that cross-species binding
occurs. At present, it appears that intracellular and cell surface
calreticulins may recognize similar carbohydrate structures, but direct
comparison of these two proteins will be required to precisely define
their ligand affinities. Interestingly, calreticulin, anchored in the
ER membrane by a genetically engineered calnexin transmembrane domain,
behaved more like calnexin than did the native non-anchored form of
calreticulin(4) . The anchored calreticulin efficiently bound
the same spectrum of nascent glycoproteins as calnexin, while the
non-anchored calreticulin did so with far less efficiency. Binding of
both forms of calreticulin required the presence of appropriate
oligosaccharide structures on the nascent glycoproteins. By analogy, we
speculate that cell surface calreticulin of mouse melanoma cells, which
we find cannot be removed by exhaustive washing (
Volume 271,
Number 14,
Issue of April 5, 1996 pp. 7891-7894
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, of 2.1 ± 0.9 
10
M
while kinetic analysis
yields an estimate of the association on rate, (K
), as 2 10
M s. The composite
results support calreticulin's lectin-like properties as well as
its proposed role in laminin recognition, both in the cell interior and
on the cell surface.
)the cell surface, perinuclear areas, and cytosolic
granules(1) . Some of these locations appear cell-specific,
that is not all cells exhibit calreticulin at each location. The ER
lumen is the most common location of calreticulin, a site where it is
found in abundance(2) . Given its strong avidity for calcium,
calreticulin has been proposed to serve as a major calcium-sequestering
protein within cells. Recently, calreticulin has been implicated as a
molecular chaperone for nascent glycoproteins(3, 4) ;
this activity resembles that of calnexin, a glycoprotein-selective
chaperone whose domain structure substantially overlaps with that of
calreticulin(5, 6) . Both chaperones appear to
selectively bind immature N-glycosyl groups of a nascent
glycoprotein in addition to binding hydrophobic regions of the protein
itself. Cell surface calreticulin also binds specific carbohydrates,
recognizing oligomannoside structures that are identical to those of
nascent glycoproteins(7, 8, 9) .Man
GlcNac
(10) ; indirect
data suggest that intralumenal calreticulin recognizes similar
structures (3, 4) . Binding studies of intact cells
show that cell surface calreticulin recognizes mannan, Man,
Man, and laminin oligomannosides but not mannose or
Man
(7, 8) . In the present study we
examine the interaction of calreticulin with glycosylated
Engelbreth-Holm-Swarm tumor laminin, which contains a repertoire of
immature to mature N-linked carbohydrates ranging from
oligomannosides to complex triantennary saccharides (11, 12, 13) , or with tunicamycin-derived
laminin, lacking such carbohydrates. Surface plasmon resonance was used
to detect and measure binding affinity; this method has the advantages
of high sensitivity and generation of real-time data(14) .
) or
without (10 mM EDTA). Proteins were coupled to the sensor chip
through free amino groups. The carboxymethylated dextran surface
(sensor chip CM5, Pharmacia) was first activated by addition of 0.2 MN-ethyl-N`-(3-diethylaminopropyl)-carbodiimide
and 0.05 MN-hydroxysuccinimide (Pharmacia amine
coupling kit), followed by addition of protein, either laminin,
unglycosylated laminin, or bovine serum albumin (BSA), at a
concentration of 20 µg/ml in 10 mM sodium acetate, pH 4.5.
Remaining N-hydroxysuccinimide esters were blocked by the
addition of 1.0 M ethanolamine hydrochloride, pH 8.5. Several
different protein concentrations were immobilized in order to optimize
conditions. In the experiments shown immobilization conditions were
controlled such that all three proteins gave approximately 3000
resonance units of immobilized material.
Immobilization of Laminin
The interaction
between calreticulin and the glycosylated and unglycosylated forms of
laminin was analyzed by the binding of soluble calreticulin to
immobilized laminin. Immobilization of laminin was accomplished by
coupling through amine groups, and successful immobilization was
confirmed by the binding of an anti-laminin antiserum (data not shown). Affinity of the Calreticulin-Laminin
Interaction
The affinity of calreticulin binding to laminin was
determined by equilibrium binding analysis on the BIAcore as has been
performed previously in other systems(16, 17) . A
range of concentrations of calreticulin was injected over the
immobilized surfaces of glycosylated laminin (Fig. 1, top), unglycosylated laminin (Fig. 1, middle),
and, as a control cell, BSA (Fig. 1, bottom). The fast
off rate of the interaction allows the binding to reach equilibrium in
a very short time; therefore short injection times were used, and very
little time was required between injections for the response to return
to base-line levels. The signal increase observed in the BSA control
cell appears to be due to refractive index changes, and this
nonspecific response was subtracted from laminin flow cells to yield
true binding responses. A plot of these data is shown in Fig. 2.
Binding of calreticulin to glycosylated laminin demonstrates
concentration dependence and saturability. A Scatchard plot of these
data is linear and gives an association constant (K
) of 2.4 10M (Fig. 3). Three independent
experiments gave a K of 2.1 ± 0.9 10M. In contrast with binding to
glycosylated laminin, binding of calreticulin to unglycosylated laminin
showed very weak affinity, typically less than 5% of that seen for the
glycosylated protein. These data indicate that calreticulin binding to
laminin is dependent on carbohydrate.
10
to 2.0 10
M was applied to three different protein surfaces. Upper panel, glycosylated laminin surface; middle
panel, unglycosylated laminin surface; lower panel,
bovine serum albumin surface. RU, resonance
units.
Calcium Is Required for the Interaction with Glycosylated
Laminin
Because calreticulin has been identified as a calcium
binding protein (18) we asked whether calcium was required for
binding to glycosylated laminin. To test this a chelating agent was
added to the calreticulin sample, and binding was tested in buffer
lacking calcium (10 mM HEPES, 150 mM NaCl, 10 mM EDTA, and 0.005% surfactant P20). The calcium-free calreticulin
was injected over the three protein surfaces, and binding responses are
shown in Fig. 4. In the absence of calcium no significant
binding is observed between calreticulin and laminin.
Analysis of the Kinetics
The off rate for this
interaction is too fast to measure accurately using this system. The
dissociation rate is clearly faster than 0.1 s. So
if one assumes a K
of 0.1 s and the association constant of 2 10M, then one can calculate an on rate (K) of 2 10M s. This is the
lowest possible estimate; it is probably faster than this. This
analysis is consistent with observations that fast-on, fast-off
kinetics is fairly typical for carbohydrate interactions (for example,
the selectins (19) ).
10
to 5.7 10M s were found,
and association constants ranging from 6.2 10
to
4.3 10
M were reported. Our results for
calreticulin binding to glycosylated laminin yield an on rate and
association constant, which differ from those values, perhaps
reflecting biological variation between various lectins and their
ligands. Given the disparate molecular sizes of calreticulin (43 kDa)
and laminin (about 900 kDa) and the magnitude of the sensorgram
signals, which reflect their binding, more than one calreticulin
molecule may bind each laminin molecule. Additional studies will be
needed to quantitatively substantiate this interpretation.
chain via surface calreticulin, thereby
stimulating cell proliferation(23) . Thus, cell signaling may
be mediated by a new class of cell surface receptors, those which have
lectin-like properties.)and is
therefore retained at the surface membrane, will differ in lectin
binding from intralumenal calreticulin. Studies are in progress to
investigate this possibility.
))
We thank Dr. B. Sutton for providing access to the
BIAcore and S. Kumar and W. Jones, Pharmacia Biosensor, for use of the
BIAcore 2000; both instruments were used for these experiments. We
thank Q. Zhu for providing unglycosylated laminin.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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